To observe the muscular protein pattern and the gut protease activity of the valuable fish species, different procedures and protocols were followed:
Sample collection: Four different commercial valuable fish species were selected as follows: Live fish (viz. rohu, catla, climbing perch and spotted snakehead) were purchased from local market (Gollamari bazaar) and kept in aquaria (climbing perch and spotted snakehead) and ponds (ruhu and catla) until needed. Upon the arrival of the fish, they were acclimatized by commercial feed (protein level 40%) in the aquaria and in the ponds. Later they were sacrificed for muscle protein and gut enzyme collection, according to Garcia-Carreno et al., (1994).
Preparation of enzyme extract: Live fish were caught from the aquaria and pond and sacrificed by dissection to collect the guts of the species. The guts of the same species were pooled together, kept at chilled condition (≤4°C) and weighed using electronic balance (Electric balance, Model-AND GF-300, Japan) and homogenized in a Potter Thomas tissue grinder with a Teflon pestle at cool temperature (≤4°C) by keeping the tissue grinder on to ice and diluted with cool distilled water (4°C) at a ratio of 1:10 W/V. The homogenate were poured into 1.5ml microfuge tubes (previously kept into ice and marked) and immediately centrifuged at 12000×g for 15 minutes at 4°C in a refrigerated centrifuge (Hettich Zentrifugen, model- D 78532, Mikro 22 R, Germany). The upper lipid layer of the supernatant, after centrifuging, was discarded. The aqueous supernatant was collected in previously cooled microfuge tubes, frozen and stored at -20°C until use (Chisty, 2005).
Preparation of protein extract: Live fish were sacrificed by dissection to collect the muscle just beneath the 1st and or 2nd dorsal fin, but above the lateral line of the respective species. The muscle of the same species were pooled together, kept at chilled condition (≤4°C) and weighted. Later, the muscle was homogenized in a Potter Thomas tissue grinder with a Teflon pestle at cool temperature (≤4°C) by keeping the tissue grinder on to ice and diluted with cool distilled water (4°C) at a ratio of 1:10 w/v. The homogenate were poured into 1.5ml microfuge tube and centrifuged at 12000×g for 15-20 minutes in a centrifuge (Eppendorf, model-5415D, Germany). The aqueous supernatant was taken in previously cooled another microfuge tube, after centrifuging. Immediately after collection of the muscle protein extract of electrophoresis was done to identify the protein pattern according to Bollag and Edelstein (2000).
Sample preparation for enzyme activity: Ten µl crude enzyme extract and 10µl sample buffer was taken, vortexed (Rocking on rotary sucker, Type 16700 Mixer, Thermoclyne) for 30 second, after vortex, the sample was ready for electrophoresis. The sample buffer was prepared according to Bollag and Edelstein (2000).
Sample preparation for protein pattern: Ten µl crude protein extract and 10µl sample buffer was taken. It was then vortexed for 30 second and heated 3-5 min at 95°C temperature in water bath (Grant instruments Ltd., Model- Hearts SG86PZ, England). Again it was vortexed and centrifuge at 12000×g for 15-20 min in a centrifuge (Eppendorf, Model- 5415 D, Germany) and was taken in a previously cool microfuge tube and was ready for electrophoresis.
Sodium Dodecyle Sulfate Polyaccrylamide Gel Electrophoresis: SDS-PAGE was done to identify the enzyme zymogram and protein pattern. Bio-Rad Mini -Gel Apparatus (Bio-Rad, MiniPROTEIN 3 cell) was used for this purpose. In brief, Twelve percent polyaccrylamide was used to prepare the linear slab gel at a dimension of 10 cm × 8 cm × 0.75 mm. In brief, the gel was prepared by pouring the separating gel in the glass slab. Stacking gel was poured onto separating gel, just after the polymerization of the separating gel. Comb (0.75 mm thickness and with 10 teeth) was inserted on it. Electrophoretic buffer was poured properly according to Bollag and Edelstein, (2000) and 10 µl sample solution was loaded onto each well. The tub was placed in a bowl containing ice to maintain the temperature below 10°C. For running the gel, 120V constant power supply was ensured (Bio-Rad, Powerpack 1000). Electrophoresis continued until the dye front migrated about to 1 cm above the bottom of the gel (around 1 hr) then Spacer (0.75 mm) was removed carefully by inserting the spacer in one corner between the plates and the gel plates were apart gently. To obtain the enzyme zymogram, the gel was soaked in 2% caesine solution (w/v) by using caesine and Tris HCl buffer, (pH 7.8) and kept at 4°C for 30 min that helped to absorb casein into the gel. Later, keeping the gel into the same solution; it was incubated for 1.5 hrs at room temperature. To obtain the protein zymogram, soaking in caesine solution was omitted. Now the gel was subjected for staining and distaining.
Gel staining: The gel was picked up and transferred to a small container, containing a small amount of coomassie gel staining solution by using coomassie Blue R-250, methanol, distilled water and glacial acetic acid and the gel, keeping into the staining solution, was agitated gently for 1-2 hrs. After staining of the gel, it was taken out from the solution, washed a few times with distilled water and was subjected to destaining.
Destaining of gel: The gel was soaked in destaining solution and kept over night with a gentle stirring. In brief the destaining solution was prepared by addition of methanol, distilled water and glacial acetic acid. Finally the gel was taken out from the solution, washed with distilled water and preserved for the zymogram.